extfieldsofstudy: Biology Medicine

text:
Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release; however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release in primary hippocampal cultures. In nonneuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of nonphotobleached probes with a time constant of ~10 s. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

Introduction
Synapses form the basis of neural communication and are exquisitely organized in facilitating point-topoint neurotransmission. Neurotransmitter release can be broadly organized into two main categories -evoked and spontaneous release. Evoked release is the more studied form of release and occurs in response to an action potential (Südhof, 2013). Spontaneous release, also referred to as activity independent release, occurs independently of action potentials. Several recent studies suggest that evoked and spontaneous release have functional segregation. For example, spontaneous and evoked release derive presynaptically from distinct synaptic vesicle pools (Sara et al., 2005), and use different release machinery as well as activate nonoverlapping sets of postsynaptic receptors (Atasoy et al., 2008;Farsi et al., 2021;Kavalali, 2015;Melom et al., 2013;Peled et al., 2014;Sara et al., 2011). Moreover, spontaneous release can activate distinct postsynaptic downstream signaling pathways compared to evoked release (Horvath et al., 2021). Spontaneous release has also been implicated in mechanisms underlying neuropsychiatric and neurological diseases as well as their treatments separate from evoked release Autry et al., 2011;Kavalali and Monteggia, 2012).
While studies have proposed a functional segregation of the two forms of release, the exact subsynaptic organization supporting spatial segregation remains unclear. Electrophysiology experiments utilizing use-dependent blockers to examine the segregation of postsynaptic receptors provide high temporal resolution of receptor activation, but do not contain spatial information about their subsynaptic location (Atasoy et al., 2008;Horvath et al., 2020). Optical imaging of fluorescent neurotransmitter probes has the advantage of reporting where neurotransmitter release occurs. For instance, wide field fluorescence imaging of fluorescent probes has examined organization of spontaneous and evoked release across different synapses (Reese and Kavalali, 2016), though it could not resolve subsynaptic sites of release. Other studies using super resolution microscopy found that trans-synaptic 'nanocolumns' aligned postsynaptic receptors and scaffolding proteins with presynaptic release sites (Tang et al., 2016), suggesting that certain proteins align with and may facilitate evoked release at spatially segregated sites. These data further support the differential organization of different modes of neurotransmitter release. Nevertheless, subsynaptic organization of different modes of release remain poorly understood as the existing tools for examining this fundamental property in real-time remain limited.
Here, we used a glutamate sensing fluorescent reporter, iGluSnFR (Marvin et al., 2013), to probe the spatial segregation of excitatory neurotransmission in hippocampal neurons. iGluSnFR is a novel probe that can resolve rapid glutamatergic transients with a high signal-to-noise ratio and is commonly used for in vivo studies (Helassa et al., 2018;Marvin et al., 2018). Using photobleaching of iGluSnFR as a tool to probe the organization of spontaneous and evoked release sites, we test whether fluorescent iGluSnFR probes within subsynaptic regions are differentially affected by photobleaching. We demonstrate that photobleaching has use-dependent properties, that iGluSnFR is a highly mobile probe expressed at synaptic surfaces, and that iGluSnFR demonstrates single synapse level resolution. Furthermore, iGluSnFR imaging can reveal the organization of distinct forms of neurotransmission at the synapse, creating a broader framework to elucidate basic principles of neurotransmission.

Results iGluSnFR localization at the plasma membrane
Primary hippocampal neuron cultures were sparsely transfected with the glutamate sensor iGluSnFR under control of a neuronal promoter (synapsin-1). Sparse labeling of neurons allows the localization of singular and nonoverlapping pre-and postsynaptic specifications ( Figure 1A).
Using confocal imaging of fixed neuronal cultures, we examined whether the expression of iGluSnFR in transfected neurons affects synapse number. We compared synapse count in iGluSnFR transfected neurons, with two control groups: GFP transfected neurons, and nontransfected neurons immunostained for a dendritic marker (MAP2). We then immunostained for pre-and postsynaptic markers (VGluT1 and PDS95, respectively), and we measured the number of colocalized pre-and postsynaptic terminals per length of neurite to estimate synapse density . We found that iGluSnFR expression did not significantly change the density of excitatory synapses (Figure 1-figure  supplement 1A, B), indicating that expression of iGluSnFR at the plasma membrane does not disrupt synaptic organization.
We next examined the resting spatial distribution of iGluSnFR probes at the plasma membrane of neurons using super resolution microscopy, which can resolve the localization of molecules past the diffraction limit of light (Huang et al., 2010). To visualize iGluSnFR via super resolution, we stained for the GFP protein in the iGluSnFR molecule. This allowed for quantification of all iGluSnFR molecules, both fluorescent (i.e., activated by glutamate) and not. After applying clustering analysis using density-based spatial clustering of applications with noise (DBSCAN) (Sawant, 2014), we observed that iGluSnFR has a greater density at synaptic regions ( Figure 1B, C). Pair correlation analysis of iGluSnFR clusters that colocalized with the postsynaptic marker PSD95 (    2A, B), revealed an increased clustering of iGluSnFR near the center of the synapse, compared to a theoretical random distribution ( Figure 1D). This suggests the existence of a general trapping mechanism at dendritic spines that confines iGluSnFR.
iGluSnFR can resolve evoked and spontaneous events at the single synapse level in hippocampal neurons Live imaging of primary neurons sparsely transfected with iGluSnFR was performed using an epifluorescence microscope equipped with an EM-CCD camera; this reduces photobleaching and toxicity during long experiments and allows resolution of fast spiking events, respectively. For this work, we focused on individual dendritic spines to isolate glutamatergic events postsynaptically. To find active synapses, we delivered a high-frequency stimulation (20 Hz, 25APs) followed by 90 mM KCl at the end of each recording, and used it to draw circular regions of interests (ROIs) of 2 µm diameter around local fluorescence maxima in dendritic spines. We then used these ROIs to analyze evoked and spontaneous activity over time ( Figure 2A). To examine whether iGluSnFR can resolve evoked transmission at single synapses, we estimated release probability (Pr), or the likelihood that a docked and primed synaptic vesicle will fuse with the plasma membrane upon depolarization from an action potential. Pr was estimated using failure analysis, where 20-40 APs at 0.2 Hz were delivered and the number of detected responses was divided by the number of stimulations. Peaks, corresponding to glutamate release events, were detected with a high signal-to-noise ratio, and peaks were included at amplitude values ≥3 standard deviations above the baseline ( Figure 2B-D). Pr has been widely reported to be low and highly variable at individual hippocampal synapses, in culture as well as in intact tissue, ranging from 0.1 to 0.2 to 0.6 (Branco et al., 2008;Chanaday and Kavalali, 2018;Farsi et al., 2021;Jensen et al., 2021;Leitz and Kavalali, 2011;Murthy et al., 1997;Tagliatti et al., 2020). Accordingly, iGluSnFR reported a low average release probability that was also highly variable among synapses ( Figure 2E-G), indicating that each ROI contained a single synapse.
To extend previous studies and validate synaptic transmission measurements using iGluSnFR, we systematically determined whether we could modulate the detected events. Pr is positively regulated by extracellular Ca 2+ levels (Chanaday and Kavalali, 2018;Leitz and Kavalali, 2011;Murthy et al., 1997). In iGluSnFR transfected neurons, perfusion of solutions with increasing Ca 2+ concentrations leads to a corresponding increase of Pr ( Figure 2E, F). Glutamate release is completely abrogated in the absence of extracellular calcium ( Figure 2F), while Pr values are shifted toward high probabilities of 0.86 ± 0.23 in 8 mM Ca 2+ , compared to 2 mM at 0.49 ± 0.32 ( Figure 2G, H). We also measured the amplitude of evoked events, which correlates to the amount of glutamate released at synapses. We found that the peak amplitudes increased at higher extracellular Ca 2+ concentrations ( Figure 2I). Based on the literature, this could be due to multivesicular release as release probabilities increase at higher Ca 2+ concentrations (Leitz and Kavalali, 2011;Rudolph et al., 2015), or glutamate spill over from adjacent sites (Armbruster et al., 2020), or a combination of these factors.
Next, we examined spontaneous neurotransmission detected by iGluSnFR. We were able to detect individual spontaneous glutamate release events at synapses with a high signal-to-noise ratio, similar to that of evoked events ( Figure 3A, B). The average frequency of spontaneous events has been reported to be approximately 0.01-0.02 Hz per synapse (Geppert et al., 1994;Leitz and Kavalali, 2014;Murthy and Stevens, 1999;Reese and Kavalali, 2016) and our detection of spontaneous events using iGluSnFR is comparable to this value ( Figure 3C). Due to the low probability of release, responses to action potentials at the level of individual synapses is a binary process: release of a single synaptic vesicle (one quanta) or a failure, as discussed above. At this scale, responses are fundamentally different from whole-cell measurements, where integrated signals from hundreds of synapses are measured. Thus, at individual synapses, both evoked and spontaneous neurotransmission lead      to release of one synaptic vesicle, thereby activating a similar numbers of probes and leading to similar event amplitudes (Leitz and Kavalali, 2011). Based on this, we expect to find similar results using iGluSnFR, and indeed we measured comparable peak amplitudes for evoked and spontaneous events ( Figure 3D). This provides further support that we are detecting the release of single synaptic vesicles at single synapses. When release properties were analyzed at the level of individual synapses, spontaneous neurotransmission rate and evoked release probability did not correlate within synapses ( Figure 3E), similar to prior studies (Leitz and Kavalali, 2014;Reese and Kavalali, 2016), thus reinforcing that evoked and spontaneous neurotransmission are partially segregated. Importantly, we observed that the spontaneous event frequency in a bath solution containing CNQX and APV (which blocks AMPAR and NMDARs, respectively) is not significantly different than in CNQX, APV, and the action potential blocker tetrodotoxin (TTX ( Figure 3F)), demonstrating that inhibition of ionotropic glutamate receptors is sufficient to suppress action potential-driven release. Furthermore, these drugs do not interact with iGluSnFR (Marvin et al., 2013). This allowed us to monitor spontaneous neurotransmission at the same neurons and synapses used for studying evoked neurotransmission, since TTX is hard to wash off. Thus, all studies on spontaneous neurotransmission using iGluSnFR were performed in the presence of only CNQX and APV.
To further evaluate the authenticity of spontaneous, action potential-independent release events detection using iGluSnFR, we next assessed the modulation of spontaneous neurotransmission by reducing extracellular Ca 2+ concentration or perfusing hypertonic sucrose ( Figure 3G). In 0 mM Ca 2+ , spontaneous release propensity is lower but not completely diminished, and iGluSnFR event amplitudes remained unchanged ( Figure 3H, I). This is consistent with previous studies demonstrating that spontaneous release is less sensitive to reductions in bath Ca 2+ than evoked release (Kavalali, 2020;Xu et al., 2009). We also perfused cells with a hypertonic sucrose (+100 mOsm) solution after recording baseline activity. Hypertonic sucrose has been shown to trigger the fusion of docked synaptic vesicles in an action potential and Ca 2+ -independent manner (Rosenmund and Stevens, 1996). While high hypertonicity (+300 to +500 mOsm) causes the whole readily releasable pool to rapidly fuse, individual release events can be resolved at lower sucrose concentrations (+100 mOsm) (Rosenmund and Stevens, 1996). Accordingly, upon perfusion of +100 mOsm hypertonic sucrose, the spontaneous event frequency detected by iGluSnFR increased, while the amplitude of detected events remained the same ( Figure 3J, K).
These results expand upon earlier work that validated iGluSnFR at single synapses (Farsi et al., 2021;Tagliatti et al., 2020), and demonstrate that iGluSnFR detects quantal release for both evoked and spontaneous neurotransmission. In these experiments, we also observed that the detection of these different modes of release can be modulated by established parameters, such as Ca 2+ and hypertonicity. These results show that iGluSnFR is a reliable and useful tool in measuring neurotransmitter release from single synapses.
iGluSnFR is a highly mobile probe in the plasma membrane but demonstrates an immobile fraction at synapses When excited by light, fluorophores can be photobleached, removing them from the normal emission cycle (McQuarrie and Simon, 1997). In contrast, fluorophores in the ground state (or nonfluorescent) cannot be photobleached, meaning that by its very nature, photobleaching is a use-dependent process ( Figure 4A). To measure the mobility of iGluSnFR at the plasma membrane, we employed fluorescence recovery after photobleaching (FRAP), which can measure fluorophore diffusion by examining the rate at which fluorescent probes replace photobleached probes within a selected region of traces from single synapse recordings (n = 10 coverslips). (E) Representative single synapse recordings in the presence of changing external Ca 2+ concentrations, as both the number and size of events increase with increasing Ca 2+ . (F) Release probability increases with increasing Ca 2+ (n = 9 coverslips). (G) Distribution of estimated release probability across single synapses in 2 mM Ca 2+ (n = 9 coverslips with a total of 636 synapses). (H) Distribution of estimated release probability across single synapses in 8 mM Ca 2+ (n = 9 coverslips with a total of 636 synapses). (I) Histogram of event sizes in the presence of increasing Ca 2+ , as well as the noise of the traces (n = 9 coverslips) Bar graphs are mean ± standard error of the mean (SEM). Significance levels were stated as follows: *p < 0.05, **p < 0.01. ns denotes nonsignificance.
The online version of this article includes the following source data for figure 2: Source data 1. Source data for Figure 1.   (Axelrod et al., 1976). Analysis was performed using a previously validated web-based software (Koulouras et al., 2018). The rate of recovery, which is a proxy for probe mobility, was estimated by fitting the curve with a single exponential equation, and the immobile fraction of probe was defined as the difference between baseline (prebleaching) and the asymptotic (postbleaching) normalized fluorescence ( Figure 4B). Using neurons transfected with iGluSnFR, we selected neuronal dendritic spines and shaft areas to bleach (ROI size = 1 µm diameter for FRAP experiments only) and monitored fluorescence recovery over time using a confocal microscope ( Figure 4C, D). In neurons, iGluSnFR is a very mobile probe, with similar recovery time constants between spine and shaft regions averaging 8.9 ± 7.0 and 9.9 ± 6.1 s, respectively ( Figure 4E). iGluSnFR also exhibited an appreciable immobile fraction of 23% and 24% in spinous and shaft regions, respectively ( Figure 4F). Immobile fractions reflect a subpopulation of probes that cannot be replenished by nonbleached probes. The existence of this immobile set of fluorophore molecules could be due to the geometry of the neuronal structure, for instance due to limitation of diffusion by surface proteins (Chen et al., 2021;Tardin et al., 2003). Another explanation could be that this is an intrinsic property of the probe, in which it interacts with proteins that tether it to the membrane surface.
To gain insight into the source of the iGluSnFR immobile fraction, we measured the mobility of the probe in a different cell system ( Figure 4G, H). We found that iGluSnFR is slightly more mobile in HEK cells (time constant of 5.6 ± 3.6 s) than in neurons ( Figure 4H, I). This suggests that iGluSnFR likely does not have an intrinsic property that leads it to tether to intracellular proteins, and that limitations in diffusion may be partially due to the geometry and asymmetric composition of neuronal membranes. In favor of this speculation, using stochastic optical reconstruction microscopy (STORM) we observed the iGluSnFR was concentrated at synapses, pointing to a putative mechanical trapping mechanism ( Figure 1). We also observed that the immobile fraction was not significantly different in HEK cells compared to neurons ( Figure 4J). Thus, the increased mobility of iGluSnFR in HEK cells compared to neurons suggests that it is less likely that there is an intrinsic property of the probe that tethers it to a specific location on the plasma membrane surface but it rather arises from neuronspecific diffusion barriers.
Photobleaching can be used as a use-dependent tool to investigate the subsynaptic spatial segregation of evoked and spontaneous neurotransmission Our results showed that iGluSnFR has single synapse level resolution, and that the probe is highly mobile within the plasma membrane with a significant immobile fraction. Based on this information, we next used photobleaching and its use-dependent properties to probe putative segregation of evoked and spontaneous neurotransmission. To do this, we measured spontaneous and evoked events in iGluSnFR transfected neurons using an epifluorescence microscope, with the same parameters used to validate single synapse neurotransmission. Synaptic ROIs were selected during high-frequency stimulation at the end of each experiment, and neuronal activity was measured retroactively. We recorded spontaneous neurotransmission for 10 min and subsequently monitored evoked release ( Figure 5A) to minimize confounding effects on spontaneous release by the electrical stimulation. We then photobleached the entire field of view with sustained maximal-intensity illumination (by removing the light dimming filter), and then resumed normal imaging of spontaneous and evoked events from all the photobleached synapses ( Figure 5A). This approach has the advantage of allowing the study of tens to hundreds of synapses, providing information about intrinsic biological variabilities. After 30 s of photobleaching, the detection of spontaneous events decreased significantly but not completely. At longer photobleaching periods of up to 20 min, spontaneous event frequency and amplitudes were decreased, although events remained detectable ( Figure 5B, C). Evoked release probability decreased significantly after 30 s. And within 5-10 min, evoked release became largely undetectable Spontaneous amplitude after perfusing Tyrode's with 100 mOsm is not significantly different (n = 8, p = 0.18). Bar graphs are mean ± standard error of the mean (SEM). Significance levels were stated as follows: ***p < 0.001, and ****p < 0.0001. ns denotes nonsignificance.
The online version of this article includes the following source data for figure 3: Source data 1. Source data for Figure 1.  Figure 5D, E), demonstrating a significant difference in its rate of photobleaching compared to spontaneous neurotransmission ( Figure 5-figure supplement 1A).
We next repeated the above set of experiments but while electrically stimulating during the photobleaching process ( Figure 5-figure supplement 1B). We did not observe a difference in evoked release detection following photobleaching with stimulation versus without stimulation   Figure 5-figure supplement 1G). One explanation could be that spontaneous release during the photobleaching period is sufficient to occlude all evoked iGluSnFR responses. To test whether a higher baseline release probability could allow more discernment of the effect of stimulating or not during photobleaching, we repeated the experiments in a higher extracellular Ca 2+ of 8 mM. In this condition, probability of release is close to 1 during baseline recordings ( Figure 2H). In agreement, we observed a higher degree of photobleaching of evoked responses when electrical stimulation was applied during the bleaching period in the presence of 8 mM extracellular Ca 2+ compared to nonstimulated neurons ( Figure 5-figure supplement 1I), indicating that the level of photobleaching of evoked responses scales up with the amount of evoked glutamate release. These results could also be explained by reduced action potential firing due to reduced excitability in the presence of high calcium (Segal, 2018).
Our results so far indicate that spontaneous neurotransmission detected using iGluSnFR is more resilient to photobleaching than its evoked counterpart. One interpretation is that iGluSnFR molecules activated by spontaneous release are in more mobile areas of the membrane and thus can be rapidly replaced by unbleached probes from other regions. If this is the case, activating all iGluSnFR molecules during the photobleaching period should abolish future detection of spontaneous neurotransmission. To test this hypothesis, we recorded spontaneous events followed by a total of 10 min of photobleaching, during which for 2 min we perfused 500 μM glutamate ( Figure 5J). We found that the detection of spontaneous iGluSnFR events was significantly and nearly completely diminished after photobleaching during glutamate perfusion ( Figure 5K). Thus, activating all iGluSnFR probes at the plasma membrane during photobleaching eliminates event detection. Importantly, this finding serves as a key negative control validating the specificity of the photobleaching approach toward bona fide glutamate release events.

Fluorescence at evoked and spontaneous release sites recover at different timescales after photobleaching
We next aimed to quantify the time course of recovery after photobleaching of spontaneous and evoked iGluSnFR events. For this purpose, we used a 10-min photobleaching period. Following photobleaching, we waited for 20 min and up to 24 hr in the absence of fluorescence imaging (i.e., in the dark), then monitored glutamatergic activity in the same neurons. Both spontaneous and evoked events were significantly bleached following the 10-min photobleaching period, although evoked events were relatively more susceptible to photobleaching confirming our previous findings. Within an hour, spontaneous events were detectable at a frequency and amplitude that was similar to that prior to photobleaching ( Figure 5H-I). On the other hand, evoked events remained undetectable an hour after photobleaching. After 8 hr, detection of evoked events resumed. At 24 hr after photobleaching, evoked event values returned to their original pre-photobleaching values ( Figure 5H, I). By plotting the event rate over time, we found that the recovery of spontaneous events occurred on the timescale of minutes, while the recovery of evoked events occurred on a longer timescale of hours. The slower recovery rate of evoked events suggests that iGluSnFR probes that respond to evoked release have less lateral mobility at the plasma membrane. This is consistent with our previous data demonstrating an immobile fraction of iGluSnFR, which may be in part due to the evoked release architecture at synapses.
In the next set of experiments, we examined whether photobleaching altered the structure and function of synapses using electrophysiological detection of glutamatergic neurotransmission. To do this, we photobleached neurons plated on gridded coverslips on the epifluorescence microscope and located the same region and neuron for electrophysiological measurements on a separate release recovering within hours Bar graphs are mean ± standard error of the mean (SEM). Significance levels were stated as follows: **p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. ns denotes nonsignificance.
The online version of this article includes the following source data and figure supplement(s) for figure 5: Source data 1. Source data for Figure 5.    Figure 6A). We recorded miniature excitatory postsynaptic currents (mEPSCs), or spontaneous neurotransmission, in both photobleached and control neurons, and observed no difference in the frequency nor the amplitude of mEPSCs between the groups ( Figure 6B). The capacitance and membrane resistance of neurons are indicative of membrane integrity and overall cell health, and these values were similar between control and photobleached neurons ( Figure 6C). Finally, we examined evoked excitatory neurotransmission ( Figure 6D), and found no difference in the amplitude of evoked postsynaptic currents, or EPSCs ( Figure 6E). There was also no difference in the paired pulse ratio, which is a proxy for release probability, between the groups ( Figure 6F). These data show that photobleaching does not affect excitatory synaptic transmission as measured by electrophysiology, nor the resting properties of neurons. Thus, the decreases seen in iGluSnFR event detection reflect the effects of photobleaching on fluorescent probes, and not intrinsic changes in neurotransmission levels due to toxic effects of prolonged illumination. (C) Capacitance and membrane resistance, markers of cell health, are not significantly different between unbleached and bleached neurons (capacitance n = 11 for both groups, membrane resistance control n = 10, photobleached n = 11). (D) Representative EPSC traces with and without photobleaching. (E) EPSC amplitudes with and without photobleaching (n = 9 for both groups). (F) Paired pulse ratios across different interstimulus intervals between neurons that have and have not been photobleached (control n = 8, bleached n = 9). Bar graphs are mean ± standard error of the mean (SEM). ns denotes nonsignificance.
The online version of this article includes the following source data for figure 6: Source data 1. Source data for Figure 6.

Discussion
In this study, we show that iGluSnFR can be used as a tool to investigate the spatial segregation of spontaneous and evoked neurotransmission. We extend the validation of iGluSnFR's ability to resolve single synapses in hippocampal neurons (Farsi et al., 2021;Tagliatti et al., 2020), creating a custom MATLAB script that can reliably detect both evoked and spontaneous release. At individual synapses, spontaneous release occurs at very low frequency; thus, it is critical to have a robust negative control. We found that spontaneous events are decreased in lower Ca 2+ concentrations. We also show that we nearly eliminate the detection of spontaneous events by photobleaching while perfusing glutamate, thus activating (and thereby making available to photobleach) all probes on the neuronal surface. These results further support the specificity of our event detection, especially spontaneous events.
Using FRAP, we established that iGluSnFR is a highly mobile probe and can replenish bleached areas within seconds, and it also possesses a sizable immobile fraction in neurons. iGluSnFR is bound to the plasma membrane by a PDGFR domain, but it is not specifically targeted to any neuronal protein; it thus theoretically should be able to freely diffuse across the entirety of the neuronal surface. We took advantage of this property of iGluSnFR and measured evoked and spontaneous release detection in varying photobleaching conditions. Photobleaching is a use-dependent process, in which only actively fluorescent probes can be bleached, and nonfluorescent probes will not be affected. While photobleaching can be an artifact of imaging that we seek to minimize, it has been used intentionally in the past -for instance, to decrease background fluorescence (Gandhi and Stevens, 2003). We employed photobleaching as a use-dependent blocker of fluorescence detection to probe synaptic physiology. When we photobleached neurons transfected with iGluSnFR, we found a differential effect on evoked and spontaneous neurotransmission, in which evoked release events photobleach much faster and more efficiently than spontaneous release events ( Figure 4B-E; note that iGluSnFR activated by tonic/ambient glutamate is also bleached - Figure 5-figure supplement 1J -). Furthermore, evoked release also takes longer to 'recover' its photobleached fluorescence when reimaged after a certain time period of time in the dark ( Figure 5A-B). Previously, pharmacological use-dependent blockers and electrophysiological techniques have been used to uncover a functional segregation between these modes of neurotransmission (Atasoy et al., 2008;Horvath et al., 2020;Peled et al., 2014;Reese and Kavalali, 2016;Sara et al., 2011). By using optical techniques, we can obtain key information about spatial location of synaptic release, as well as indirectly probe the location of presynaptic glutamate release by examining glutamate responses at the postsynapse.
Our results build upon earlier work that show evoked release may be localized to specific and subsynaptic regions. For instance, Tang et al., 2016 found that pre-and postsynaptic proteins align in nanocolumns that preferentially allow evoked release to occur. In agreement with this finding, a recent study identified LRRTM2 as a trans-synaptic adhesion protein that regulates AMPAR positioning at release sites (Ramsey et al., 2021). Several studies have demonstrated a mobile and immobile fraction of AMPARs (Chen et al., 2021;Opazo and Choquet, 2011), which may coincide with the immobile fraction of iGluSnFR found in our studies. Collectively, these studies suggest that certain proteins align to facilitate evoked release within spatially restricted regions.
Our findings also revealed a consistent subsynaptic structure in which evoked release is more clustered and restricted than spontaneous release, as such an organization would lead to photobleaching having a stronger effect on evoked release. Photobleached probes within the evoked release site have greater difficulty diffusing out of the bleached region and allowing unbleached probes to enter. Thus, within a few minutes, evoked release reaches a nearly undetectable level, and it takes a much longer time for the signal to recover. In contrast, spontaneous release may occur more broadly across the synapse, likely with less pronounced surface protein alignment at the site of release (Figure 7). These events are harder to photobleach because the mobility of iGluSnFR is high, such that by the time another spontaneous event occurs, unbleached iGluSnFR will have replenished the photobleached probes and be able to respond to subsequent spontaneous release events. iGluSnFR moves faster, on the order of seconds, than the rate of photobleaching which occurs on the order of minutes. Another explanation could be that there is a greater number of spontaneous release sites compared to evoked release sites, so that even if one release site is bleached, there are many others that have not been affected and could still fluoresce upon glutamate release. However, the latter proposal is inconsistent with the observation that 10-15 min long application of the use-dependent N-methyl-D-aspartate receptor (NMDA) receptor blocker MK-801 or folimycin, a use-dependent blocker of

Model Schematic
Photobleaching Immediately after photobleaching Minutes after photobleaching, there is some spontaneous event "recovery" Hours after photobleaching, there is some evoked event "recovery" . Model schematic demonstrating the clustered and diffusion restricted organization of evoked events, compared to the more diffusely located and freely moving structure of spontaneous event sites. Evoked events are more readily photobleached, likely due to its clustered location and less diffusible surface. Spontaneous events are still detectable after photobleaching, likely due to the more freely diffusible nature of its structure and their Figure 7 continued on next page presynaptic vesicle acidification, are sufficient to suppress spontaneous neurotransmission (Atasoy et al., 2008;Sara et al., 2005). Therefore, our photobleaching period of 20 min is beyond this time period and should have been sufficient to suppress all events if photobleached iGluSnFRs were not rapidly replenished. Finally, the time for evoked iGluSnFR events to recover fluorescence takes longer than for spontaneous events, which further suggests that there is a greater diffusion barrier within evoked release sites compared to spontaneous sites. Our experiments were performed in an in vitro culture system as much prior synaptic work has been done in primary cultures, thus our results more directly build upon previous work (Ramsey et al., 2021;Renner et al., 2017;Tang et al., 2016;Zhang et al., 2018). While we would expect the findings from primary cultured neurons to largely translate to an intact brain system, there may be some differences. For instance, the differences in extracellular matrix composition may affect diffusional properties, and the 3D nature of synapse organization within the dense neuropil could lead to differences in glutamate dynamics compared to a monolayer of neurons (Matthews et al., 2022). It is also possible that more complex ex vivo brain tissues may introduce technical complications in detecting differences in photobleaching and fluorescence recovery of iGluSnFR probes. Furthermore, imaging synapses in intact brain systems would be better performed on a two-photon microscope, and this would require a recharacterization of iGluSnFR dynamics and photobleaching properties by a different microscope.
The differences in event detection after photobleaching and fluorescence recovery, as well as a clustered distribution uncovered by super resolution analysis, support a model of synaptic organization where different modes of neurotransmission are segregated. Here, we demonstrate that photobleaching can be used to probe the spatial segregation of different modes of neurotransmission. These results demonstrate a novel use for a widely recognized property of fluorescence probes, expanding the ways in which we use photobleaching as a molecular tool, as well as demonstrating the spatial organization of different modes of neurotransmission.

Materials and methods
dispersed location. Recovery for spontaneous release occurs on the order of minutes, likely due to synaptic and extrasynaptic diffusion of unbleached probes into the bleached regions. Recovery for evoked release occurs on the order of hours.

Figure 7 continued
Continued on next page

Continued
Primary dissociated hippocampal neuron culture preparation Primary hippocampal cultures were generated by dissecting hippocampi from P1-3 Sprague-Dawley rats as previously described (Kavalali et al., 1999), with some modifications. Briefly, dissected hippocampi were washed and treated with 10 mg/ml trypsin and 0.5 mg/ml DNAse at 37°C for 10 min.
Tissue was washed again, dissociated with a P1000 tip, and centrifuged at 1000 rpm for 10 min at 4°C. Cells were then resuspended and plated on Matrigel-coated 0 thickness glass coverslips in 24-well plates at a density of four coverslips per hippocampus. Cultures were kept in humidified incubators at 37°C and gassed with 95% air and 5% CO 2 . Plating media contained 10% fetal bovine serum (FBS), 20 mg/l insulin, 2 mM L-glutamine, 0.1 g/l transferrin, 5 g/l D-glucose, 0.2 /g NaHCO 3 in minimal essential medium (MEM). After 24 hr, plating media was exchanged for growth media containing 4 μM cytosine arabinoside (as well as 5% FBS, 0.5 mM L-glutamine, and B27) to inhibit glial proliferation. On days in vitro (DIV) 4, growth media was exchanged to a final concentration of 2 μM cytosine arabinoside. Cultures were then kept without disruption until DIV 14-21.

Sparse neuron transfection
Neuronal transfections were performed on DIV 7 using a calcium phosphate kit (ProFection Mammalian Transfection System, Cat # E1200, Promega), based on a previously described method (Sando et al., 2019). Briefly, A precipitate was formed by mixing the following per each well in a 24-well plate: 1 μg of plasmid DNA, 2 μl of 2 M CaCl 2 , and 13 μl dH 2 O. This mixture was then added dropwise to 15 μl of 2× N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid (HEPES), while vortexing between drop addition. The precipitate was allowed to form for 15 min. Neuron conditioned media was saved and replaced with MEM and 30 μl of plasmid mixture was added dropwise to each well. Plates were returned to 5% CO 2 incubator at 37°C for 30 min. Then cells were washed twice with MEM, after which previously saved conditioned media was added back to each well. Neurons were analyzed at DIV 15-17 using a confocal microscope.
To isolate excitatory currents, 50 μM APV and 50 μM picrotoxin (PTX, ionotropic GABA receptor inhibitor) were added to the bath solution. To isolate mEPSCs, 1 μM TTX sodium channel inhibitor, 50 μM PTX, and 50 μM D-AP5 were added. For EPSC recordings, the field stimulation was provided using a parallel bipolar electrode (FHC) immersed in the external bath solution, delivering 35 mA pulses (0.1 ms duration) via a stimulus isolation unit. Miniature events were identified with a 5-pA detection threshold and analyzed with MiniAnalysis (Synaptosoft, Fort Lee, NJ, USA).

Live fluorescence imaging
Imaging experiments were done in Tyrode's buffer (as described above). Tyrode's solution containing either 0, 2, or 8 mM Ca 2+ with osmolarity titrated to 310-320 mosM, and 50 μM APV and 10 µM CNQX to prevent recurrent neuronal activity. Fluorescence was recorded using a Nikon Eclipse TE2000-U inverted microscope equipped with a ×60 Plan Fluor objective (Nikon, Minato, Tokyo, Japan), a Lambda-DG4 illumination system (Sutter Instruments, Novato, CA, USA) with FITC excitation and emission filters, and an Andor iXon + back illuminated EMCCD camera (Model no. DU-897E-CSO-#BV; Andor Technology, Belfast, UK). Images were acquired at 50 Hz to resolve fast spiking glutamatergic peaks. To induce photobleaching, the neutral density filter within the LAMDA-DG4 illumination system was removed in order to use 100% light intensity. This filter was reintroduced for subsequent live imaging of neurotransmisison after photobleaching.
Spontaneous activity was recorded over the course of 6-10 min. Evoked responses were elicited using a parallel bipolar electrode, delivering 35 mA pulses (0.1ms duration) at 5-s intervals. At the end of each experiment, presynaptic boutons were visualized by delivering a high-frequency electrical stimulation (25 Hz 20 action potentials) or by perfusing 90 mM KCl in Tyrode's solution.

Fluorescence analysis
Images were analyzed using Fiji (Schindelin et al., 2012). Local fluorescence maxima during 90 mM KCl stimulation were located using a custom macro and used to draw circular ROIs (of 2 μm diameter) around synapses. Fluorescence intensity over time was measured for each ROI and exported to Excel, along with the image metada containing treatment and stimulation time information. Data were analyzed using an unbiased method based on our previous studies (Chanaday and Kavalali, 2018). Briefly, background was subtracted linearly, and traces were smoothed at every three or five points. Spontaneous events were detected using a threshold of 3 standard deviations (SDs) above a moving average (baseline) of 4 s. Evoked events detection was time locked within 0.3 s of an AP delivery, at a threshold of 3 SD above baseline. Parameters including frequency, release probability, and amplitude were automatically estimated. All custom Matlab (Mathworks, Natick, MA, USA) scripts are available on Github (https://github.com/camilleswang/iGluSnFR-Analysis, copy archived at swh:1:rev:8f529b-60ce0561ad771dd553cc4f477dbbf7cfaf; Wang, 2021) and upon request.

Fluorescence recovery after photobleaching
FRAP experiments were performed on an LSM 510 META confocal microscope (Carl Zeiss, Oberkochen, Germany) with a ×63 (NA1.4) objective. A small region ~1 μm in diameter was selected to be bleached. An unbleached reference region was selected to correct for artifactual photobleaching, and a background region was selected for normalizing the signal. Photoleaching was performed over four to five scans at 100% laser power. Fluorescence recovery data were run through easyFRAP-web (https://easyfrap.vmnet.upatras.gr/), a web-based tool for the analysis of FRAP data, which provides photobleaching depth, gap ratio, normalization data, and curve fitting parameters (Koulouras et al., 2018). Values were computed with a double normalization method (using a neighboring unbleached area as control) and a single exponential equation curve fitting. Immobile fractions were calculated from the asymptote of the single exponential equation (for detailed info please see Koulouras et al., 2018).

Immunofluorescence
At DIV 16-17, neuron cultures were fixed with 4% paraformaldehyde (PFA) and 4% sucrose in phosphate-buffered saline (PBS) at room temperature for 15 min. After three washes, cells were permeabilized for 30 min with 0.2% Triton-X in PBS. Following another three washes, blocking solution consisting of 1% bovine serum albumin (BSA) and 2% goat serum was added for 1-2 hr. Primary antibody diluted in blocking solution was added and incubated overnight at 4°C in a humid chamber. Primary antibody against MAP2 (1:500) was used to detect neuronal architecture, anti-vGluT1 (1:500) was used to detect presynaptic boutons, and anti-PSD95 (1:200) was used to detect postsynaptic specifications. The following day, coverslips were washed three times then incubated with speciesappropriate Alexafluor secondary antibodies at 1:500 for 60-90 min at room temperature. Coverslips were then washed and mounted on glass slides, and they were imaged using an LSM 510 META confocal microscope (Carl Zeiss, Oberkochen, Germany) with a ×63 (NA1.4) objective.

Super resolution microscopy
Neuronal cultures were grown on 1.5 glass bottom Mattek dishes (Cat # P35G-1.5-14-CGRD), and they were transfected with iGluSnFR at DIV7. At DIV 16-17, neuron cultures were fixed and permeabilized similar to immunofluorescence experiments. Cells were then washed and blocked with 1% BSA, 2% goat serum, and 2% donkey serum for 2 hr at room temperature. Primary antibody against GFP (rabbit, Synaptic Systems, 1:300) was added to detect iGluSnFR molecules overnight, as well as PSD95 (mouse, Synaptic Systems, 1:100). The following day, primary antibody was washed off and cells were incubated in secondary antibody (AlexaFluor 647 at 1:500; AlexaFluor 568 at 1:500) for 1.5 hr at room temperature. Secondary antibodies were then washed off and cells were postfixed with 4% PFA for 10 min to enable long-term storage of samples in PBS.
For STORM, the extracellular imaging buffer was made fresh on ice prior to imaging (50 mM MEA, 1× glucose oxidase, 1× catalase, 4% 2-mercaptoethanol in Buffer B, which is comprised of 50 mM Tris-HCl + 10 mM NaCl + 10% glucose). TetraSpeck beads (100 nm; Invitrogen) mounted on a glass coverslip were used to calibrate alignment between the two channels. Imaging was performed on a Vutara VXL Microscope from Bruker. All data analysis was performed with the Vutara software, using the spatial distribution module and cluster analysis module to measure density values and detect clustering of iGluSnFR tagged by GFP. The Vutara software finds the geometrical center of the cluster to run a pair correlation analysis.

Statistical analysis
Data in graphs were presented as mean ± standard error of the mean (SEM) unless indicated otherwise. Sample sizes were stated in the figure legends and represented as the number of coverslips, unless otherwise indicated. Statistics were done on the averages of coverslips, rather than individual synapses to avoid falsely significant results due to very large sample sizes (the number of synapses and release events can run in the thousands). Individual synaptic values are represented as denoted in several graphs to demonstrate the distribution of values. Sample sizes were based on previous studies in the field of molecular and cellular neuroscience as opposed to using statistical methods prior to experimentation. To ensure reproducibility, each set of experiments were performed across multiple coverslips in at least two sets of cultures. Vutara Software Analysis was used to perform the pair correlation analysis of the super resolution images using the spatial distribution module and cluster analysis module, as well as to detect clustering of iGluSnFR tagged by GFP. GraphPad Prism was used to perform the statistical analyses of all other sets of experiments.
A Welch's t-test was used to compare effects in pairwise datasets obtained from synapses or neurons under distinct conditions. A Kolmogorov-Smirnov test was used to compare the cumulative histogram of two groups. A chi-squared test was used to analyze the correlation between two groups. For parametric analysis of multiple comparisons, two-way analysis of variance (ANOVA and one-way ANOVA) with Tukey post hoc analysis were used. Outliers were identified with Robust regression and Outlier removal (ROUT) method. Differences among experimental groups were considered statistically significant when a p value ≤0.05 was reached. Specifics of statistical tests and p value denotations are listed in figure legends.


s2fieldsofstudy: Biology Medicine

text:
Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome

Congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency is an autosomal recessive disorder. The 21-hydroxylase enzyme P450c21 is encoded by the CYP21A2 gene located on chromosome 6p21.33 within the HLA major histocompatibility complex. This locus also contains the CYP21A1P, a non-functional pseudogene, that is highly homologous to the CYP21A2 gene. Other duplicated genes are C4A and C4B, that encode two isoforms of complement factor C4, the RP1 gene that encodes a serine/threonine protein kinase, and the TNXB gene that, encodes the extracellular matrix glycoprotein tenascin-X (TNX). TNX plays a role in collagen deposition by dermal fibroblasts and is expressed in the dermis of the skin and the connective tissue of the heart and skeletal muscle. During meiosis, misalignment may occur producing large gene deletions or gene conversion events resulting in chimeric genes. Chimeric recombination may occur between TNXB and TNXA. Three TNXA/TNXB chimeras have been described that differ in the junction site (CH1 to CH3) and result in a contiguous CYP21A2 and TNXB gene deletion, causing CAH-X syndrome. TNXB deficiency is associated with Ehlers Danlos syndrome (EDS). EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders. As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB-gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

INTRODUCTION
Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive enzymatic disorders, caused by a deficiency of one of the enzymes required for cortisol biosynthesis in the adrenal cortex. CAH is mostly associated with pathogenic variants in the 21-hydroxylase (CYP21A2) gene (1,2). Residual enzyme activity defines the clinical severity of the disease. The most common form of CAH is 21-hydroxylase deficiency (21-OHD) accounting for 95% of cases. Prevalence of the most severe or classic forms is 1:16,000 live births in the Caucasian population, while the non-classic or late-onset form is the most common, with a prevalence between 1:1000-1:500 live births depending on ethnicity and geographic area (1)(2)(3).
The CYP21A2 gene is located on the long arm of chromosome 6, within the major human histocompatibility complex (HLA), a region with a complex gene organization (4-7) (Figure 1). There is a nonfunctional pseudogene (CYP21A1P), located approximately 30 kb from the CYP21A2 gene. Both the functional gene and the pseudogene comprise ten exons that share a 98% nucleotide sequence identity (4)(5)(6)(7). The close proximity and high level of homology between the functional and the non-functional gene facilitates misalignment resulting in recombination events that frequently produce large CYP21A2 gene deletions and conversions as well as point pathogenic variants in the CYP21A2 gene. The locus, is one of the most complex in the human genome since it contains three other genes, RP1 also called STK19 that encodes a serine/threonine protein kinase, C4A and C4B that encode two isoforms of complement factor C4, and TNXB that encodes an extracellular matrix glycoprotein tenascin-X (TNX), as well as two pseudogenes, RP2 and TNXA, that together constitute a 30kb genetic unit called the RCCX module (4)(5)(6)(7). The genetic diversity of the RCCX module is highly attributable to nonallelic homologous recombination (NAHR). Unequal crossover during meiosis generates large structural rearrangements and copy number changes, whereas gene conversion mediates relatively short sequence transfers (8).
The TNXA and TNXB genes lie on the opposite strand of DNA from the other genes of the cluster and therefore have the opposite transcriptional direction. The last exon of TNXA and TNXB partially overlap the 3′ untranslated region of exon 10 in CYP21A1P and CYP21A2, respectively (9). TNXB is a large gene composed of 44 exons spanning 68.2 kb, whereas TNXA is a truncated gene of 4.5kb, homologous to exons 32 to 44 of TNXB.
As mentioned above, chimeric CYP21A1P/CYP21A2 genes are caused by homologous recombination between CYP21A2 and its pseudogene CYP21A1P as a result of unequal crossover and are found in 20-25% of alleles in CAH due to 21-OHD. To date, nine different CYP21A1P/CYP21A2 genes have been characterized (10).
The unequal crossover may, in some cases, produce TNXA/ TNXB chimeras from which the CYP21A2 gene is completely removed (Figure 1). At present three different TNXA/TNXB chimeras have been described-CH1, CH2 and CH3-that differ in the junction site. CH1 is characterized by a 120-bp deletion in exon and intron 35 carried over from TNXA pseudogene sequences leading to haploinsufficiency (11). CH2 lacks this deletion but contains a pseudogene-derived variantc.12174C>G (p.Cys4058Trp)-in TNXB exon 40 producing the loss of a critical disulfide bond in the tertiary structure of the TNX C-terminal fibrinogen-like domain (12). CH3 is characterized by the presence of any variant of a cluster of three pseudogene-derived variants -exon 41: c.12218G>A, (p.Arg4073His); exon 43: c.12514G>A (p.Asp4172Asn) and c.12524G>A (p.Ser4175Asn)-. The cluster of these three pseudogene-derived variants may differ in the haplotypes found in the three chimeras and they do not always cosegregate. Modeling and energy calculations suggest that the p.Arg4073His variant is detrimental to proper TNX folding while the remaining variants in the cluster did not significantly affect the folding energies in the models (13) . In addition, some CH1 haplotypes that harbor the 120-bp deletion in exon 35 but lack the p.Cys4058Trp variant in exon 40 have been found. This may be explained by the fact that the allele frequency of pseudogene derived-variants is not 100 percent. Since CH2 and CH3 produce altered proteins rather than reducing TNX expression, they are associated with a dominant-negative effect. The TNX protein belongs to a family of evolutionarily conserved large glycoproteins of the extracellular matrix. It plays a role in collagen deposition by dermal fibroblasts and is expressed in FIGURE 1 | Genomic organization of the CYP21 locus on chromosome 6p21.1-21.3: The functional CYP21A2 gene and its non-functional CYP21A1P pseudogene are arranged in tandem repeat with the two C4 genes that encode factor four of the complement system, the serinethreonine nuclear protein kinase active gene RP1 (STK19) and a truncated pseudogene RP2 (STK19P). The functional TNXB and the TNXA pseudogene are located on the complement strand. Schematic representation of a 30-kb deletion as a result of unequal crossover during meiosis and the formation of the three described TNXA/TNXB chimeras (CH1, CH2 and CH3), which differ in the junction site. CH1 is characterized by a 120-bp deletion in exon and intron 35 carried over from TNXA pseudogene sequences. CH2 lacks this deletion but contains a pseudogene-derived variant c.12174C>G the dermis of the skin and in the connective tissue of the heart and skeletal muscle. TNXB deficiency leads to Ehlers-Danlos Syndrome (EDS) and up to 10% of CAH patients also have EDS, an entity called CAH-X. EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility (14).
As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

MOLECULAR ANALYSIS OF THE TNXB GENE
The first report of TNX deficiency was a description of a patient with CAH and EDS (15). In 2001, Schalkwijk et al. reported a subtype of EDS that is now known as classic-like EDS (clEDS) (16). clEDS is an autosomal recessive form of EDS and is caused by a deficiency of TNX encoded by the TNXB gene. The authors evaluated 151 patients with the classic hypermobility or vascular types of EDS, together with 168 patients with other conditions (psoriasis and rheumatoid arthritis) and 21 healthy individuals for the presence of TNX and tenascin-C by enzyme-linked immunosorbent assay. The patients were tested for the 30-kb deletion leading to a TNXA/TNXB chimeric gene by allelespecific PCR and for other point mutations by sequencing the coding sequence of the TNXB gene. Four of five TNX-deficient patients were identified to have homozygous TNXB mutations. Subsequently, the same authors reported an association between haploinsufficiency of the TNXB gene and the hypermobility-type EDS (hEDS) in 20 heterozygous family members; however, generalized joint hypermobility (GJH) was observed in only nine female patients (17).
The first evaluation of the potential clinical implications of TNXB heterozygosity in CAH patients was reported by Merke et al. in a large prospective observational study from the National Institutes of Health (NIH) (11). One hundred ninety-three unrelated patients with CAH were evaluated clinically for manifestations of EDS and genetically for TNXB mutations. DNA was analyzed for the presence of a contiguous gene deletion syndrome caused by deletion of CYP21A2 and its flanking gene TNXB by PCR multiplex ligation-dependent probe amplification (MLPA) and confirmed by Southern blot. This deletion generated a TNXA/TNXB chimera characterized by a 120-bp deletion in exon 35 of the TNXB gene, which is replaced by TNXA sequences. In addition, TNXB sequencing was performed in a group of patients with one or more joint or skin manifestations. Heterozygosity for the TNXB deletion was observed in 7% of CAH patients, who were considered to have CAH-X syndrome. Here, the association between the hypermobility phenotype and TNXB-gene haploinsufficiency was established. In 2015, the same authors identified a pseudogene-derived variant-c.12174C>G (p.C4058W)representing a novel TNXA/TNXB chimera that did not involve a 120-bp deletion in exon 35 in seven families with CAH-X (12). Interestingly, this variant did not affect the protein expression of tenascin in dermal fibroblasts and for this missense variant a dominant-negative mechanism was proposed, which is different from the haploinsufficiency caused by the abovedescribed chimera. Of 246 CAH probands screened, 14 carried previously described TNXA/TNXB chimeras (CH1) while seven unrelated patients carried the novel TNXB variant (CH2) resulting in a prevalence of CAH-X of 8.5% (12). The same authors later reported three patients with biallelic CAH-X and identified a novel dominant-negative chimera (CH3) characterized by any of three TNXB variants [exon 41: c.12218G>A (p.Arg4073His); 191 exon 43: c.12514G>A (p.Asp4172Asn) and c.12524G>A (p.Ser4175Asn)] (Figure 1). This study presented evidence for disrupted TNX function, since by computational data the p.Arg4073His variant was predicted to reduce protein-folding energy by interfering with a cation-pi interaction between p.Arg4073 and p.Phe4080 (13).
Molecular analysis of the TNXB gene is challenging due to the presence of the pseudogene, which makes next-generation sequencing highly complicated in these cases. In 2019, Lao et al. reported a high-throughput CAH-X screening method based on allele-specific PCR to assess the copy numbers of TNXB exons 35 and 40. The method is compatible with either quantitative PCR or droplet digital PCR and allows detection of CH1 and CH2. Using this methodology, the authors found a 15.6% prevalence of CAH-X, which was higher than previously estimated. The prevalence was especially high (29.2%) in subjects with a 30-kb deletion genotype (18).
In 2020, Gao et al. assessed the prevalence of the chimeric TNXA/TNXB gene and clinical symptoms in a Chinese cohort of 424 21-OHD patients. MLPA analysis and Sanger sequencing was performed to genetically identify the CAH-X syndrome. In this cohort, 14% of the patients with 21-OHD were found to have a chimeric TNXA/TNXB gene (19). Finally, also in 2020, our group reported the molecular TNXB-gene status and clinical evaluation of the EDS phenotype in a cohort of 337 Argentine 21-OHD patients to assess the prevalence of this condition in our population. TNXB gene analysis was performed in 66 unrelated CAH patients that were carriers of the 30-kb CYP21A2 gene deletion. A molecular strategy based on MLPA and Sanger sequencing analysis was developed for the detection of the three previously described TNXA/TNXB chimeras (20). The overall prevalence of CAH-X in 21-OHD patients in our cohort was 14%, which was similar to that previously found in the large cohort from the NIH and in the Chinese population (15% and 14%, respectively). In our population of 21-OHD patients carrying the 30-kb CYP21A2 gene deletion in which the junction site was downstream exon 7 both in the homozygous or the heterozygous state, the incidence of TNXA/ TNXB chimeras was 73% (48/66), similar to the prevalence of 62.8% (59/94) found in the Chinese population. On the other hand, in the NIH cohort a prevalence of 29.2% (21/72) was reported. The reason for the lower prevalence found in the latter study is that the authors reported the presence of TNXA/TNXB chimeras in CAH patients that were carriers of all types of 30-kb CYP21A2 deletions described.
In addition to TNXA/TNXB chimeras, pathogenic variants in the TNXB gene have been described as a less frequent cause of TNX deficiency. Pathogenic variants were detected in the coding region of the EGF-like repeats, the fibronectin type III domain or C-terminal domain structurally related to fibrinogen of the TNX protein. Moreover, recently a splice donor site variant has been described as a cause of the hEDS type (21). Finally, the variable prevalence of CAH-X reported in the different cohorts might be related to the molecular strategies used.

CLINICAL MANIFESTATIONS OF CAH-X PATIENTS
CAH-X patients are reported to have a wide range of connective tissue abnormalities, including generalized joint hypermobility, subluxations, chronic arthralgias, soft or velvety skin, mild skin hyperextensibility, and variable systemic manifestations. The severity of the phenotype may be correlated with the dosage of the dominant alleles, as monoallelic CAH-X is associated with hEDS, the mildest and most common EDS type, and biallelic CAH-X with the more severe clEDS subtype.
Biallelic CAH-X patients resemble the clEDS type, with extreme joint laxity, with or without recurrent joint dislocations, and skin hyperextensibility with a velvety skin texture and absence of atrophic scarring. Easily bruisable skin and soft-tissue injuries as well as organ prolapse, pes planus, piezogenic papules, chronic pain, arthralgias, and cardiac abnormalities have been described. Thus far, 12 patients with biallelic CAH-X syndrome have been reported (13,15,16,19,20); however, clinical information is not available for all cases. Our group reported four biallelic CAH-X patients (two with a CH1/CH1 and two with a CH1/CH2 combination) (20). Both CH1/CH2 patients had a more severe EDS phenotype, with greater skin involvement. Nevertheless, the low number of homozygous patients reported to date limits the possibility to draw robust conclusions based on these data. In addition, in the latter patients cardiac defects were detected; one had an atrial septal and the other a mild pulmonary valve defect. None of the patients developed either atrophic scarring, organ prolapse, or any other complications during the 3 years of follow-up; however, these observations are limited by the young age of our patients and the short-term follow-up. Chen et al. reported three biallelic patients of 14, 19, and 29 years of age, all displaying unique combinations of TNXB variants in both alleles; one of them was homozygous for CH2/CH2, the other was a CH2/CH3 compound, and the third a CH2/CH1 compound (13). All of them had skin hyperextensibility and significant joint hypermobility. Joint laxity was extreme and two patients had a history of joint dislocations, chronic arthralgias, and chronic tendinitis and/or bursitis. Unlike our findings, the authors reported widened atrophic scarring, rectal prolapse, severe gastroesophageal reflux, high palate, and elongated uvula in all three biallelic patients. Mild ventricular enlargement was detected in two patients. Currently, the limited number of cases reported and the heterogeneous combination of TNXB variants they display make it difficult to establish a certain genotype/phenotype correlation.
Long-term follow-up is needed to specifically evaluate quality of life in CAH-X patients. On the other hand, clEDS patients (without CAH) are affected by soft-tissue fragility and long-term complications (13,15,16,19,20).
Most patients with the monoallelic form of CAH-X syndrome present with the clinical spectrum of hEDS with variable expression at different stages of life, predominantly characterized by GJH, mild skin hyperextensibility, and soft ve lve ty sk in, w ith ou t ab nor m a l s ca r r ing. Re la te d musculoskeletal complications, such as recurrent joint dislocations, pes planus, and chronic arthralgias, have been reported. Other associated features include functional gastrointestinal alterations and cardiac disorders. Although this type of EDS may cause chronic pain and reduced quality of life, life-threatening complications are uncommon (11,12,15,17,19,20).
The underlying chimera translates into different degrees of hEDS phenotypes. Compared to TNXB haploinsufficiency caused by CAH-X CH1, a dominant negative effect related to CAH-X CH2 causes a more severe phenotype with increased joint and skin manifestations (12,20). Gastrointestinal disorders, such as chronic gastroesophageal reflux and irritable bowel syndrome, hernias, and organ prolapse, are also more frequently reported in patients that are heterozygous for CAH-X CH2 than in those with CAH-X CH1. Data on the phenotype associated with the less frequent CAH-X CH3 are scarce (12,19,20,(22)(23)(24). Recently, Gao et al. reported the presence of CH3 in 11 patients; however, clinical information was available for only one of them, who had joint hypermobility and poor wound healing (19). In our cohort, CH3 was only found in one monoallelic patient who was not available for clinical evaluation (20).
Although the exact role of CAH-related hormonal factors and/or chronic glucocorticoid treatment in connective tissue dysplasia is not yet completely understood, it has been shown that CAH-X patients are consistently more severely affected than patients with homozygous or heterozygous TNX-deficient-type EDS without CAH (11,12,16,19,25).
Furthermore, a phenotype that is varied and usually milder than that of monoallelic CAH-X patients has been reported in the relatives of CAH-X patients, who were carriers of one CAH-X allele, but not affected by CAH. Some carriers were observed to have less joint, heart, or gastrointestinal symptoms, while others were asymptomatic (12,13,17,19,20). On the other hand, connective tissue dysplasia has been described in CAH patients without demonstrated TNXB deficiency (11,12).
Recently, Lao et al. reported a novel cause of CAH-X syndrome, not associated with pathogenic TNXA/TNXB chimeras but due to a TNXB splice donor site variant (21). As currently TNXB testing remains challenging, at least in the routine diagnostic approach, the diagnosis of CAH-X still relies on clinical evaluation.
Connective tissue dysplasia should be evaluated in all CAH patients, especially in those harboring a deletion in the CYP21A2 gene. Screening for GJH and other soft tissue features presents age-related difficulties and should therefore be adapted to age, systematically evaluated, and retrospectively asked for. The Beighton Score (shown in Figure 2) remains the most objective assessment tool to measure GJH (22,26).
Knowledge of the TNXB status in CAH will not only offer patients a better understanding of their symptomatology with the burden of the diagnosis of a second genetic disease, but also assures specific clinical management with a focus on preventing musculoskeletal manifestations and complications.
Cardiovascular alterations may be underreported in CAH-X patients, probably related to the difficulties patients without cardiac symptoms may have to access specific diagnostic studies, such as cardiac magnetic resonance imaging (MRI), outside the context of a research study protocol, as observed in different series. A variable prevalence of cardiac abnormalities was found in different studies using different diagnostic methods.
Currently, no specific medical or genetic therapies are available for CAH-X patients. Management consists of interdisciplinary medical care, rehabilitation, and monitoring of major and organ-specific complications (24,27,28). Further TNXB and CAH-X studies are necessary to define detailed surveillance guidelines for these and other long-term complications and to develop prevention strategies.

CONCLUSIONS
Different studies on the CYP21A2 gene have improved our knowledge on TNXB-related disorders. The TNXB gene encodes an extracellular matrix glycoprotein named TNX. EDS may be due to TNXB deficiency and up to 10% of 21-OHD CAH patients also have CAH-X. Chimeric recombination of the TNXB and TNXA genes may occur, and three TNXA/TNXB chimeras that differ in the junction site (CH1 to CH3) resulting in a contiguous CYP21A2 and TNXB gene deletion, named CAH-X syndrome, have been described. Molecular studies are the gold standard to assess the presence of TNXA/TNXB chimeras. On the other hand, molecular analysis of the TNXB gene is challenging due to the presence of a pseudogene and next generation sequencing is highly complicated in these cases. For this reason, among others, TNX-deficient type EDS may be underdiagnosed. The variable prevalence of CAH-X reported in different cohorts may be related to the molecular strategies applied. Systematic study of TNXB status in individuals with a previous diagnosis of CAH and carriers of the complete 30-kb deletion of CYP21A2 is highly recommended. Moreover, molecular genetic testing of CAH-21OHD should include TNXA/TNXB chimera analysis (29).
EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility as well as cardiovascular alterations. Cardiac disorders, in particular heart valve abnormalities, may be underdiagnosed, probably because of the young age of the majority of reported CAH-X patients and EDS-related cardiac abnormalities may appear with aging. In addition, it is unlikely that patients without heart symptoms are routinely checked for cardiac abnormalities with echocardiogram and/or cardiac MRI and currently only data from patients participating in specific CAH-X research studies are available. Severity of the phenotype may be correlated with the dosage of the dominant alleles, as monoallelic CAH-X patients have the mildest and most common EDS type and biallelic CAH-X patients the more severe clEDS subtype.
CAH-X patients are consistently more severely affected than patients with homozygous or heterozygous TNX-deficient type EDS without CAH; however, the impact of the hormonal milieu on TNX and its role in connective-tissue pathophysiology is still poorly understood. In this line, carriers of one CAH-X allele who are not affected with CAH have a varied and milder EDS phenotype than monoallelic CAH-X patients.
Once the diagnosis of CAH-X has been established, it is advisable to guarantee long-term follow-up of these patients by medical specialists with a focus on preventing musculoskeletal manifestations and complications.
Finally, in order to prevent long-term musculoskeletal disorders, timely diagnosis of CAH-X is important and physical therapy for joint instability is recommended. In addition, molecular characterization of CAH-X is relevant for genetic counseling.

ETHICS STATEMENT
Written informed consent was obtained from the participant's legal guardians for the publication of any identifiable material in this study.

AUTHOR CONTRIBUTIONS
All authors contributed equally to design of the manuscript. RM, AM, and AB wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.